During the previous grant period, we found that topical, intradermal application of cowhage spicules induces itch by non-histaminergic mechanisms. Electrophysiological recordings from unmyelinated (C-fiber) and myelinated (A-fiber) afferents in peripheral nerves revealed that histamine and cowhage activate different peripheral pathways. For C-fibers, mechano-insensitive afferents (MIAs) were activated by histamine but not cowhage, whereas mechanosensitive nociceptive afferents (MSAs) were responsive to both, but the responses to cowhage were substantially greater. Similarly, histamine but not cowhage activated A-fiber MIAs, whereas A-fiber MSAs responded to cowhage or histamine or both. Again, the responses to cowhage were significantly greater than the responses to histamine. These findings indicate that the sensation of itch can be mediated through multiple afferent pathways. The proposed electrophysiological experiments will help to determine the relative roles of these different classes of afferents in signaling the different sensations associated with the pruritic stimuli. Specifically, we propose: 1) To determine whether pruriceptive and/or nociceptive selective classes of MIAs exist by investigating the responsiveness of A- and C-fiber MIAs to pruritic and algesic stimuli. 2) To investigate a potential peripheral mechanism for scratch induced itch relief or itch relieve by application of thermal stimuli. We will determine if application of such stimuli leads to a decrement in the response to pruritic stimuli of A-fiber and C-fiber MSAs. Preliminary results reveal significant fatigue in the responses of C-MSAs to cowhage after mechanical stimulation. 3) To investigate the differential chemosensitivity of nociceptive and pruriceptive small-diameter afferents using a novel application technique, i.e. heat-inactivated, cowhage-spicules shown in project 1 to cause itch and pain when loaded with chemicals including capsaicin, histamine and non-histaminergic pruritic agents such as cysteine proteases. We postulate that different populations of small myelinated and unmyelinated MSAs mediate the sensation of itch and pain produced by these stimuli. The results from these studies will enhance our understanding of the peripheral neuronal apparatus underlying the sensation of itch and pain. RELEVANCE (See Instructions): Itch that is resistant to treatment, for example with antihistamines, is a major clinical problem. The proposed studies will increase our understanding about how the sensation of itch is mediated in the nerve fibers of the skin. The findings of these studies may potentially lead to the development of better strategies for the treatment of itch.